The present invention is directed to a biological or biotransformation process for producing 11.alpha.-hydroxyl derivatives of 17.beta.-N-mono-substituted carbamoyl-4-azasteroid compounds using the green algae, Selenastrum capricornutum.
The art reveals that a number of microorganisms selected from fungi and bacteria have been used in biotransformation processes to produce biotransformed products. U.S. Pat. No. 4,431,736 describes the production of phenylhydroquinone from biphenyl by biotransformation with selected fungi. This reference specifically describes the microbiological oxidation of biphenyl to bis-hydroxlated biphenyl employing the fungal organism, Thamnostylum piriforme.
U.S. Pat. No. 4,397,947 describes a microbial process for 9.alpha.-hydroxylation of steroids using the organism, Nocardia canicruria. It is known that the microorganisms Achromobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium, Microbacterium, Micybacterium, Protaminobacter, Serratia, and Streptomyces may grow on steroid compounds and degrade the ring system in a non-specific or specific manner. See U.S. Pat. Nos. 4,320,195; 4,175,006; 4,035,236; 4,029,549; 5,004,695. Nocardia species have also been used to hydroxylate non-steroidal compounds. See U.S. Pat. Nos. 4,537,859; 4,582,804. U.S. Pat. No. 2,602,769 describes the 11-hydroxylation of a steroid compound using the fungal microorganism, Rhizopus sp.
The art also reveals that certain undesirable physiological manifestations including acne vulgaris, seborrhea, female hirsutism, male pattern baldness and benign prostatic hypertrophy are the result of hyperandrogenic stimulation induced by excessive accumulation of testosterone or similar androgenic hormones. Early attempts to provide chemotherapeutic agents to counter the unwanted physiological effects of hyperandrogenicity resulted in the discovery of several steroidal anti-androgens that unfortunately had hormonal side effects. For example, the estrogens counteract the effects of androgens but also have a feminizing effect. Non-steroidal anti-androgens such as 4'-nitro-3'-triflouromethylisobutylanilide have also been developed. See Neri et al., Endo., 91, (2), (1972). These compounds may be peripherally active and compete for androgenic receptor sites which could feminize a male host or the male fetus of a female host.
It more recently became known in the art that the princial mediator of androgenic activity in some target organs is 5.alpha. dihydrotestosterone which is formed locally in the target organ with the catalytic assistance of the enzyme testosterone-5.alpha.-reductase. It therefore has been postulated and demonstrated that inhibitors of testosterone-5.alpha.-reductase prevent or diminish the symptoms of hyperandrogenism. See Nayfe et al., Steroids, 14, 269 (1969) and Voight et al., Endocrinology, 92, 1216 (1973). It has also been demonstrated that topical application of either testosterone or 5.alpha.-dihydrotestosterone causes enlargement of the female hamster flank organ, an androgen dependent sebaceous structure. However, concommitant administration of 4-androsten-3one-17beta-carboxlyic acid or its methyl ester inhibited the response elicited by testosterone but did not inhibit the response elicited by 5.alpha.-dihydrotestesterone. These results indicated that these particular compounds were antiandrogenic because they inhibited testosterone-5.alpha.-reductase. Since this discovery, a number of steroidal 5.alpha.-reductase inhibitors have been found. See, U.S. Pat. Nos. 5,032,586; 5,026,882; 5,017,568; 4,970,205; 4,970,204; and 4,954,446.
A number of steroidal derivatives with a hydroxyl functionality at the 11 position (either alpha or beta) are known. See, for example, U.S. Pat. Nos. 4,361,558; 4,430,270; 4,432,905; 4,446,072; and 4,448,725. Steroidal derivative(s) with a hydroxyl functionality at the 11 position which inhibit 5.alpha.-reductase are known. See U.S. Pat. No. 5,041,433.
A number of 4-aza steroid compounds are known. See, for example, U.S. Pat. Nos. 2,227,876; 3,239,417; and 3,264,301; French Pat. No. 1,465,544; Doorenbos et al., J. Pharm. Sci., 62, (4), 638-640 (1973); J. Pharm. Sci., 60(8), 1234-1235 (1971); J. Pharm Aci., 63(4) 620-622 (1974).
In addition, U.S. Pat. Nos. 4,377,584; 4,220,775; 4,859,681; and the articles J. Med. Chem., 27, 1690-1701 (1984) and J. Med. Chem., 29, 2998-2315 (1986) by Rasmussen et al. and U.S. Pat. Nos. 4,845,104 and 4,732,897 disclose 4-aza-17.beta.-substituted- 5.alpha.-androstan-3-ones which are said to be useful in the treatment of hyperandrogenic conditions. Several other U.S. patents refer to aza-steroids including U.S. Pat. Nos. 5,061,802 (17.beta.-aminibenzoyl-4-aza-5.alpha.-androst-1-en-3-ones) and 5,049,562 (17.beta.-Acyl-4-aza-5.alpha.-androst-1-ene-3-ones).
U.S. Pat. No. 4,760,071 specifically refers to the compound 17.beta.(N-t-butylcarbamoyl)-4-aza-5.alpha.-androst-1-en-3-one. Heretofore, however, the art has not referred to any oxidized 4-azasteroids produced via a biotransformation process using the green algal organism Selenastrum capricornutum nor to a biological process for the production of the novel compound 17.beta.(N-t-butylcarbamoyl)-11.alpha.(hydroxyl)-4-aza-5.alpha.-androst-1- en-3-one, which has been found to be an inhibitor of testosterone prostatic 5-.alpha.-reductase. The compounds described in this invention are produced via biotransformation in the ubiquitous green algae, Selenastrum capricornutum. Use of this organism as an environmental barometer of pollutants is known. See Eirkson et al., "Environmental Assessment Technical Assistance Handbook", Government Publication No. PB87-175345, National Technical Information Service (1987). There is a need, however, for a process that converts steroidal compounds to oxygenated or hydroxylated compounds. This invention claims and describes use of a green algal microorganism to oxidize or hydroxylate steroidal compounds.